hBDNF原核表达载体的构建及其表达

Construction of expression vector for hBDNF fusion gene and its expression in E.coli

目的:利用基因重组技术构建人脑源性神经营养因子(human brain-derived neurotrophic factor,hBDNF)的原核表达载体pBV220/mhBDNF,诱导表达融合基因,鉴定BDNF的生物学活性。方法:制备重组质粒pGEM-T/hBDNF及hBDNF的成熟肽基因片段,经限制性内切酶联合酶切,将hBDNF亚克隆到原核表达载体pBV220,诱导表达克隆基因,培养鸡胚背根神经节,检测表达蛋白的活性。结果:重组原核表达质粒pBV220/mhBDNF构建正确,表达框架未发生改变。成功诱导表达了融合基因并成功地分泌表达hBDNF蛋白。表达蛋白组可见大量神经突起自神经节组织块外周缘向四周呈放射状长出,损伤组未见明显神经突起长出。结论:成功构建了原核表达载体pBV220/mhBDNF,重组质粒可分泌表达hBDNF蛋白,而且hBDNF蛋白有良好的生物活性。本实验为进一步开展hBDNF基因治疗感音神经性耳聋提供了可能。

Objective:To construct the prokaryotic expression vector for fusion gene pBV220/mhBDNF and evaluate the bioactivity of BDNF.Methods:hBDNF and mature hBDNF were recombined in phage vector pGEM-T Easy,Then it was subcloned into prokaryotic expression vector pBV220,then called pBV220/hBDNF.The plasmid was induced to express in E.coli at 42℃.The secreted and expressed protein was analysied by SDS-PAGE.0.1ml expression hBDNF protein was addecd to dissociated spinal cord-dorsal root ganglion(SC-DRG).The test and ...